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Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene

  • Background The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment. Results Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that thisBackground The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment. Results Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.show moreshow less

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Metadaten
Author:Wenhao Yan, Dijun Chen, Kerstin Kaufmann
DOI:https://doi.org/10.1186/s13007-016-0125-7
ISSN:1746-4811
Parent Title (English):Plant methods
Publisher:BioMed Central
Place of publication:London
Document Type:Article
Language:English
Year of first Publication:2016
Year of Completion:2016
Publishing Institution:Universität Potsdam
Release Date:2016/05/24
Tag:Germline transmission; Large fragment deletion; Multiplex mutagenesis; RNA-guided Cas9
Volume:12
First Page:1
Last Page:9
Funder:Universität Potsdam, Publikationsfonds
Grant Number:PA 2016_20
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
5 Naturwissenschaften und Mathematik / 58 Pflanzen (Botanik) / 580 Pflanzen (Botanik)
Peer Review:Referiert
Grantor:Publikationsfonds der Universität Potsdam
Publication Way:Open Access
Licence (German):License LogoCreative Commons - Namensnennung, 4.0 International
Notes extern:Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe ; 224